Methods and compositions for altering mucus secretion

ABSTRACT

Methods and compounds for increasing or decreasing mucus secretion in subjects, and particularly mucus secretion in the airways, are described. Methods of screening compounds for the ability to increase or decrease mucus secretion are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/914,020, filed Dec. 31, 2001 now U.S. Pat. No. 7,265,088, and claimspriority under 35 U.S.C. §371 from PCT Application No. PCT/US00/05050(published under PCT Article 21(2) in English), filed on Feb. 24, 2000,which claims the benefit of U.S. application Ser. No. 09/256,154, filedFeb. 24, 1999, abandoned, the disclosures of which are incorporated byreference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to methods and compositions that areuseful in regulating mucus secretion, and that may be utilized intreating medical conditions where it is desirable to increase ordecrease mucus secretion.

BACKGROUND OF THE INVENTION

Mucus is a biological liquid that is capable of forming gels. It is amixture of components, including water and secretory products from avariety of cells. Expectorated human airway mucus contains approximately95% water and 5% solids; the solids contents include 2-3% proteins andglycoproteins, 1% lipids, and 1% minerals. See Boat et al., Biochemistryof Mucus, In: Airway Secretion, Takashima and Shimura (eds.), MarcelDekker, 1994. Mucins, also called mucous glycoproteins or epithelialglycoproteins, are glycoconjugates characterized by numerousoligosaccharide side chains linked to a peptide core by N- andO-linkages.

In the airways, mucins are released onto the airway surface from gobletcells on the surface epithelium, and from mucus cells of submucosalglands. The total amount of surface liquid (mucus) in the airways is theresult of the rate of mucus secretion in conjunction with the rate ofclearance of mucus (by epithelial reabsorption, evaporation, ciliarytransport, and cough transport). Under “normal” conditions, the rate ofsecretion and clearance of mucus are balanced so that only a thinsurface layer of liquid covers the tracheobronchial tree. Mucushypersecretion (if not accompanied by a concomitant increase in mucusclearance) results in accumulation of airway mucus, which can result inairflow obstruction and increased retention of inhaled particulatematter and microbial matter. Existing strategies to reduce luminal mucusin the airways include inhibition of mucus hypersecretion using indirectpharmacological action, changing the physical characteristics of mucusto enhance ciliary action, and enhancement of cough clearance of mucus.

Hypersecretion of mucus contributes to the pathogenesis of a largenumber of airway inflammatory diseases in both human and non-humananimals. Increased mucus secretion is seen in chronic disease statessuch as asthma, COPD and chronic bronchitis; in genetic diseases such ascystic fibrosis; in allergic conditions (atopy, allergic inflammation);in bronchiectasis; and in a number of acute, infectious respiratoryillnesses such as pneumonia, rhinitis, influenza or the common cold.Accordingly, new methods and therapeutic compounds able to decrease orlessen mucus secretion are desirable.

Under-secretion of mucus also has harmful effects. Airway mucus acts asa physical barrier against biologically active inhaled particles, andmay help prevent bacterial colonization of the airways and inactivatecytotoxic products released from leukocytes. King et al., Respir.Physiol. 62:47-59 (1985); Vishwanath and Ramphal, Infect. Immun. 45:197(1984); Cross et al., Lancet 1:1328 (1984). In the eye, mucus maintainsthe tear film, and is important for eye health and comfort. Mucussecretion in the gastrointestinal tract also has a cytoprotectivefunction. The role of mucus as a chemical, biological and mechanicalbarrier means that abnormally low mucus secretion by mucous membranes isundesirable.

In view of the foregoing, improved methods and compositions able toalter (i.e., increase or decrease) mucus secretion from epithelial cellsand mucus membranes are desirable.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of inhibiting mucussecretion by a mucus-secreting cell, by administering to the cell amucus-inhibitory amount of a compound that inhibits MARCKSprotein-related mucus secretion.

A second aspect of the present invention is a method of inhibiting mucussecretion by a mucus-secreting cell, by administering to the cell apeptide inhibitor of MARCKS-related mucus secretion.

A third aspect of the present invention is a method of inhibiting mucussecretion in the airways of a subject in need of such treatment, byadministering to the airways of the subject a mucus-inhibiting amount ofa compound that inhibits the MARCKS-related release of mucin.

A fourth aspect of the present invention is a method of increasing mucussecretion by a mucus-secreting cell, by administering to the cell asecretion-enhancing fragment of a MARCKS protein in an amount sufficientto increase mucus secretion by the cell, compared to that which wouldoccur in the absence of the protein fragment.

A fifth aspect of the present invention is a method of inhibiting mucussecretion by a mucus-secreting cell, by administering to the cell amucus-inhibiting amount of an antisense construct that specificallybinds to endogenous MARCKS protein encoding sequences underphysiological conditions, wherein mucus secretion by the cell isinhibited compared to that which would occur in the absence of suchadministration.

A sixth aspect of the present invention is a pharmaceutical formulationcontaining a mucus-inhibiting peptide fragment of MARCKS and apharmaceutically acceptable carrier.

A seventh aspect of the present invention is an oligonucleotideconsisting of about 10 to 50 nucleotides having a nucleotide sequencethat hybridizes to nucleotide molecules encoding a MARCKS protein underphysiologic conditions. The oligonucleotide inhibits expression of theMARCKS protein when administered to a cell containing the endogenousnucleotide molecules.

An eighth aspect of the present invention is a method of inhibitingmucus secretion by a mucus-secreting cell, by administering to the cella mucus-inhibitory amount of a compound that binds to a target siteselected from (a) mucin granule membranes at the site bound by MARCKSprotein; and (b) MARCKS protein at the mucin granule binding site. Theamount of mucus secreted by the cell is reduced compared to that whichwould occur in the absence of the compound.

A ninth aspect of the present invention is a method of enhancing mucussecretion by a mucus-secreting cell, by administering to the cell amucus-enhancing amount of a compound that binds to an endogenousinhibitor of MARCKS protein.

A tenth aspect of the present invention is a method of screening a testcompound for the ability to bind, in a mucus-secreting cell, to a siteselected from (a) mucin granule membranes at the site bound by MARCKSprotein, and (b) a MARCKS protein at the mucin granule membrane bindingsite. The method comprises administering the test compound to amucus-secreting cell and then detecting whether the test compoundinhibits binding of endogenous MARCKS protein to the mucin granulemembrane.

An eleventh aspect of the present invention is a method of enhancingmucus secretion by a mucus-secreting cell, by administering to the cella mucus enhancing amount of a compound that increases the amount ofMARCKS protein in the cell, such that mucus secretion is enhanced incomparison to that which would occur in the absence of the compound.

A final aspect of the present invention is the use of a compound asdescribed above (including proteins, peptide fragments,oligonucleotides, and non-peptide compounds) that may be used to altermucus secretion for the preparation of a medicament for the secretion ofmucus in a subject in need thereof, as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of stimulated mucin secretion by human bronchialepithelial cells in vitro in response to varying amounts of the MANSpeptide (SEQ ID NO:1). Column 1=media/control (no peptide, nostimulation); column 2=100 nM PMA and 1 μM 8-Br-cGMP (stimulatedsecretion); column 3=1 μM MANS peptide, 100 nM PMA and 1 μM 8-Br-cGMP;column 4=10 μM MANS peptide, 100 nM PMA and 1 μM 8-Br-cGMP; and column5=100 μM MANS peptide, 100 nM PMA and 1 μM 8-Br-cGMP. Single asterisks(*) indicate that the measured response was statistically different thanthe media control (column 1), and double asterisks (**) indicate thatthe response was statistically different than that of stimulated cellsthat were not exposed to the MANS peptide (column 2).

FIG. 1B graphs the inhibition of basal mucin secretion by humanbronchial epithelial cells exposed to the MANS peptide (SEQ ID NO:1) ora negative control peptide consisting of the same amino acids as theMANS peptide, but in random order (RNS peptide; random N-terminalsequence). Column 1=media control; column 2=one hour incubation with 100μM of RNS; column 3=one hour incubation with MANS peptide. The singleasterisk (*) indicates that the response in column 3 was statisticallydifferent than the media control (column 1).

FIG. 2 is a graph of the effects of varying amounts of the MANS peptide(SEQ ID NO: 1) on UTP-induced mucin secretion by human bronchialepithelial cells in vitro. Column 1 is the media/control; column 2=0.1mM UTP; column 3=0.1 mM UTP and 1 μM MANS peptide; column 4=0.1 mM UTPand 10 μM MANS peptide; and column 5=0.1 mM UTP and 100 μM MANS peptide.Single asterisks (*) indicate that the measured response wasstatistically different than the media control (column 1), and doubleasterisks (**) indicate that the response was statistically differentthan that of UTP-stimulated cells that were not exposed to the MANSpeptide (column 2).

FIG. 3A is a graph of the effects of the MANS peptide (SEQ ID NO:1) andthe MA-PSD peptide (SEQ ID NO:2) on stimulated mucin secretion by humanbronchial epithelial cells in vitro. Column 1=media/control; column2=100 nM PMA; column 3=100 μM PMA and 1 μM 8-Br-cGMP; column 4=100 nMPMA, 1 μM 8-Br-cGMP and 1 μM MA-PSD peptide; column 5=100 nM PMA, 1 μM8-Br-cGMP and 10 μM MA-PSD peptide; column 6=100 n™ PMA, 1 μM 8-Br-cGMPand 100 μM MA-PSD peptide; column 7=100 nM PMA, 1 μM 8-Br-cGMP and 1 μMMANS peptide; column 8=100 nM PMA, 1 μM 8-Br-cGMP and 10 μM MANSpeptide; and column 9=100 nM PMA, 1 μM 8-Br-cGMP and 100 μM MANSpeptide. Single asterisks (*) indicate that the response wasstatistically different than the media control (column 1), and doubleasterisks (**) indicate that the response was statistically differentthan that in stimulated cells that were not exposed to the MANS peptide(column 3).

FIG. 3B graphs the effect of one hour of incubation with the MA-PSDpeptide (SEQ ID NO:2) on basal mucin secretion by human bronchialepithelial cells in vitro. Column 1=media/control; column 2=100 μMMA-PSD; column 3=10 μM MA-PSD; column 4=1 μM MA-PSD. The single asterisk(*) indicates that the response in column 3 was statistically differentthan the media control (column 1).

FIG. 4 is an illustration of a proposed signaling pathway ofMARCKS-mediated mucin secretion by human epithelial cells. In thisFigure, PKC=protein kinase C; PKG cGMP-dependent protein kinase;GC-S=soluble guanylyl cyclase; PP2A=protein phosphatase 2A; NO=nitricoxide; GTP=guanosine triphosphate; and cGMP=cyclic guanosinemonophosphate. In this proposed pathway, mucin secretagogues (shown inthe Figure as binding to a receptor) interact with airway epithelial(goblet) cells and activate two separate protein kinases: PKC and PKG.Activated PKC phosphorylates MARCKS, causing its translocation from theplasma membrane to the cytoplasm, where it is targeted to the mucingranule membrane with the assistance of MARCKS-associated proteins. PKG,activated via the nitric oxide (NO)-cGMP-PKG pathway, in turn activatesa cytoplasmic protein phosphatase 2A (PP2A), which dephosphorylatesMARCKS, thus stabilizing its attachment to the granule membrane andallowing MARCKS to cross-link actin filaments. This tethers the granuleto the cytoskeleton for movement and exocytosis.

FIG. 5 is a graph of the effects of a MARCKS antisense oligonucleotideon stimulated mucin secretion by human bronchial epithelial cells invitro. Column 1=media/control; column 2=cells stimulated with 100 nM PMAand 1 μM 8-Br-cGMP; column 3=5 μM control oligonucleotide, 100 nM PMAand 1 μM 8-Br-cGMP; column 4=5 μM antisense oligonucleotide, 100 nM PMAand 1 μM 8-Br-cGMP. Single asterisks (*) indicate that the measuredresponse was statistically different than the media control (column 1),and double asterisks (**) indicate that the response was statisticallydifferent than that observed in stimulated cells that were not exposedto the any oligonucleotide (column 2).

FIGS. 6A, 6B, and 6C together illustrate that TNF-α up-regulates MARCKSexpression and augments mucin hypersecretion. NHBE cells were incubatedwith 10 ng/ml human recombinant TNF-α or medium alone for 4 hrs, thenstimulated with PMA (100 nM)+8-Br-cGMP (1 μM) for 15 min, or UTP (0.1mM) for 2 hrs. Secreted mucin was collected and measured by ELISA. TotalRNA and protein were isolated from treated cells. MARCKS mRNA wasassessed by Northern hybridization, and MARCKS protein by the WesternBlot technique.

FIG. 6A is a Northern-blot and graph indicating an increase in MARCKSmRNA in cells incubated with TNF-α (lane 2 of blot, column 2 of graph)compared to cells incubated in medium alone (lane 1 of blot, column 1 ofgraph).

FIG. 6B is a Western-blot and graph showing a three- to four-foldincrease in MARCKS protein in cells incubated with TNF-α (lane 2 ofblot, column 2 of graph) as compared with cells incubated with mediumonly (lane 1 of blot, column 1 of graph).

FIG. 6C is a graph showing that in cells incubated with TNF-α, mucinhypersecretion was significantly augmented in response to subsequentstimulation by PMA+8-Br-cGMP or UTP when compared to mucin secretion ofcells incubated in medium only. Data are presented as mean±SEM (n=6 ateach point). Single asterisks (*) indicate a statistically significantdifference from control (medium-only) samples (p<0.05). Single crossmarks (†) indicate a statistically significant difference from stimulus(p<0.05).

FIG. 7 is a graph showing that okadaic acid, a phosphatase inhibitor,blocks mucin hypersecretion induced by PMA+8-Br-cGMP or UTP. NHBE cellswere pre-incubated with okadaic acid (500 nM) for 15 min at 37° C./5%CO₂, then stimulated with PMA (100 nM)+8-Br-cGMP (1 μM) for 15 min, orwith UTP (0.1 mM) for 2 hours. Secreted mucin in the apical medium wascollected and assayed by ELISA. Column 1 shows the results of incubationwith medium alone for 30 min. Column 2 shows the results ofpre-incubation with medium alone for 15 min, then incubation withPMA+8-Br-cGMP for an additional 15 min. Column 3 shows the results ofpre-incubation with okadaic acid for 15 min, then co-incubation withPMA+8-Br-cGMP for an additional 15 min. Column 4 shows the results ofincubation with medium alone for 2 hrs. Column 5 shows the results ofpre-incubation with medium alone for 15 min, then incubation with UTPfor an additional 2 hrs. Column 6 shows the results of pre-incubationwith okadaic acid for 15 min, then co-incubation with UTP for anadditional 2 hrs. Data are presented as mean±SEM (n=6 at each point).Single asterisks (*) indicate a statistically significant differencefrom control (p<0.05). Single cross marks (†) indicate statisticallysignificant difference from stimulus (p<0.05).

FIG. 8 is a graph showing that mucin hypersecretion induced by UTPinvolves activation of PKC and PKG. NHBE cells were pre-incubated withthe indicated inhibitor (described below) for 15 min, then stimulatedwith UTP (0.1 mM) for 2 hours. Secreted mucin in the apical medium wascollected and assayed by ELISA. Column 1 indicates the results ofincubation with medium alone. Column 2 indicates the results ofincubation with 0.1 mM UTP. Column 3 indicates the results of incubationwith 0.1 mM UTP+500 nM calphostin C (an inhibitor of PKC). Column 4indicates the results of incubation with 0.1 mM UTP+10 μMRp-8-Br-PET-cGMP (an inhibitor of PKG). Column 5 indicates the resultsof incubation with 0.1 mM UTP+50 μM LY83583 (an inhibitor of solubleguanylyl cyclase). Column 6 indicates the results of incubation with 0.1mM UTP+500 nM KT5720 (an inhibitor of PKA). Data are presented asmean±SEM (n=6 at each point). Single asterisks (*) indicate astatistically significant difference from control (p<0.05). Single crossmarks (†) indicate a statistically significant difference from UTPstimulation (p<0.05).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Mucus is the clear viscous secretion of the mucous membranes, andcomprises water, mucin, lipids, and various inorganic salts. Mucin is acarbohydrate-rich glycoprotein that is secreted by specializedepithelial cells (known as goblet cells), the submaxillary glands, andother mucous glandular cells. Goblet cells are epithelial cellsspecialized for secretion and containing an accumulation of mucoussecretory granules.

Mucous tissue (or mucosa) lines various anatomic structures in themammalian and avian body, including the eyes, respiratory tract(alveoli, bronchi, oral cavity, larynx, nasal cavity, pharynx, trachea),gastrointestinal tract (esophagus, stomach, small and large intestine,rectum), and genitourinary tract (urethra, urinary bladder, uterus andvagina).

Alterations in the quantity of mucus secretions may be due to variousunderlying factors, including a change in the amount of mucousglycoproteins secreted from mucus-secreting cells, a change in the totalnumber of mucus-secreting cells, or combinations thereof. Mediatorsreleased by the inflammatory response are known to act as mucussecretagogues, including lipid mediators, oxygen metabolites, and othercell-specific products. Larivee et al., In: Airway Secretion, Takishimaand Shimura (Eds.), Marcel Dekker Inc., 1994, pages 469-511.

As used herein, “hypersecretion” of mucus refers to production of mucusabove a normal or basal amount, or production of mucus in an amount thatleads to pathological changes or symptoms.

As used herein, the “inhibiting mucus secretion” refers to a lesseningor reduction in mucus secretion; it is not meant to imply the completecessation of mucus secretion. A treatment that inhibits mucus secretionresults in decreased mucus production compared to that which wouldoccur, or would be expected, in the absence of such treatment.

Amounts of mucus secreted by a cell in culture, or by a tissue in vivocan be measured or assessed using methods as are known in the art.

As used herein, the “enhancement” or “stimulation” of mucus secretionrefers to an increase in mucus secretion. A treatment that enhancesmucus secretion results in increased mucus production compared to thatwhich would occur, or would be expected, in the absence of suchtreatment.

As used herein, “stimulated mucus secretion” refers to mucus secretionthat occurs in response to a secretagogue; this is contrasted to “basalmucus secretion” that occurs under normal physiological conditions.

As used herein, a compound that inhibits the MARCKS protein-mediatedrelease of mucin granules (or mucus) includes those compounds that actupon a step in the MARCKS protein-mediated signaling pathway thatresults in mucus secretion, causing a reduction in mucus secretion.

As used herein, “endogenous” refers to compounds that are naturallyoccurring in a cell. Endogenous MARCKS protein thus refers to MARCKSprotein that is found within a cell, as opposed to MARCKS proteinintroduced into that cell (either administered directly or by geneticengineering techniques).

As used herein, an “active fragment” of a MARCKS protein is one thataffects (inhibits or enhances) the MARCKS protein-mediated release ofmucus that occurs in response to a secretagogue such as UTP (uridine5′-triphosphate). An active peptide fragment of MARCKS comprises anamino acid sequence that is identical or substantially identical to acontiguous sequence of amino acids found in a naturally occurring MARCKSprotein. Active MARCKS protein fragments are typically at least aboutfive, ten, fifteen, twenty or twenty-five amino acids in length, but areshorter than the complete MARCKS protein. Active MARCKS proteinfragments preferably have fewer than about fifty, seventy-five, onehundred or two hundred amino acids.

A “mucus inhibitory” or “mucus inhibiting” amount of a compound is thatamount which reduces or inhibits mucus secretion, compared to that whichwould occur in the absence of the compound. A “mucus enhancing” amountof compound is that amount which enhances or increases mucus secretion,compared to that which would occur in the absence of the compound. Forexample, as described herein, peptides of SEQ ID NO:2 were found toincrease mucus secretion in airway epithelium in vivo when provided in acertain amount, and to inhibit mucus secretion when provided in greateramounts. The most effective amount of a particular peptide will varydepending upon the peptide, route of administration, and condition beingtreated. As used herein, the term “compound” is to be broadly construedto include proteins, peptide fragments, nucleotides, oligonucleotides,and other non-protein chemicals.

As used herein, a peptide inhibitor of MARCKS-related mucus secretion(or release of mucin) is a peptide that, when provided to a mucussecreting cell, inhibits or reduces the secretion of mucus compared tothat which would occur in the absence of said peptide.

As used herein, a peptide enhancer of MARCKS-related mucus secretion (ormucin release) is a peptide that, when provided to a mucus secretingcell, enhances or increases the secretion of mucus compared to thatwhich would occur in the absence of said peptide.

As used herein, “oligonucleotide” refers to DNA or RNA and can includesense and/or antisense strands as appropriate to the desired effect.Oligonucleotides useful in the present invention may be incorporatedinto recombinant expression vectors that include a promoter and othersequences necessary for expression of the desired translation products(such as a peptide). Alternatively, ‘naked’ oligonucleotides may bedelivered to target cells, as is known in the art (see, e.g., Felgner etal., U.S. Pat. No. 5,580,859).

Mucosa or mucous membranes, as used herein, refers to mucosal tissues ofa host wherever they may be located in the body including but notlimited to respiratory passages (nasal, oral, tracheal, bronchial),genital passages (vaginal, cervical, anal and penile), urinary passages(urethra, bladder), and the eyes.

The present invention provides methods and compositions that are usefulin inhibiting mucus secretion from epithelial cells. The presentinventors have determined that mucin secretory processes in epithelialcells involve the protein kinase C (PKC) substrate MARCKS protein(myristolated alanine-rich C-kinase substrate). By blocking orinhibiting the function and/or production of MARCKS protein in secretoryepithelial cells, mucin secretion is reduced over that which wouldotherwise occur (i.e., that would occur in the absence of suchtreatment). The present invention also provides methods and compositionsthat are useful in enhancing mucus secretion from epithelial cells. Byenhancing the function and/or production of MARCKS protein in secretoryepithelial cells, mucin secretion is increased over that which wouldotherwise occur (i.e., that would occur in the absence of saidblocking).

The present inventors have shown that use of a fragment of the MARCKSprotein reduces mucus secretion by epithelial cells. Additionally, useof antisense fragments directed against the MARCKS mRNA sequence alsohas been shown to decrease mucus production in epithelial cells.

Despite the previous identification of numerous mucus secretagogues,common signaling pathways and intracellular molecules involved in mucinsecretion have not previously been elucidated. The present inventionexploits the unexpected discovery that the myristolated alanine-richC-kinase substrate (MARCKS) protein is involved in the secretory processof cells, and particularly in the secretion of mucus from epithelialcells (such as goblet cells). MARCKS protein is a major cellularsubstrate for protein kinase C (PKC), and the present inventors' studiesindicate that it is a central, convergent molecule controlling releaseof mucin granules. While not wishing to be held to any single theory ofthe present invention, the MARCKS-related secretion of mucus appears toinvolve the interaction of mucin secretagogues with airway epithelial(goblet) cells and the activation of two separate protein kinases: PKCand PKG. Activated PKC phosphorylates MARCKS, causing its translocationfrom the plasma membrane to the cytoplasm, where it is targeted to themucin granule membrane with the assistance of MARCKS-associatedproteins. PKG, activated via the nitric oxide (NO)-cGMP-PKG pathway, inturn activates a cytoplasmic protein phosphatase 2A (PP2A), whichdephosphorylates MARCKS, stabilizing its attachment to the granulemembrane and allowing MARCKS to cross-link actin filaments, therebytethering the granule to the cytoskeleton for movement and exocytosis.This proposed signaling pathway is generally depicted in FIG. 4.

The present inventors identified MARCKS mRNA and protein in humanbronchial epithelial cells, and both mRNA and protein levels increasedwith secretory cell differentiation in vitro. The MARCKS in these cellswas phosphorylated by the phorbol ester PMA (phorbol 12-myristate13-acetate), while subsequent addition of a cGMP activator(8-bromo-cGMP), caused dephosphorylation. Mucin secretion provoked(i.e., stimulated) by the pathophysiologically relevant secretagogueuridine triphosphate (UTP) (or by a combination of PMA and 8-bromo-cGMP)was inhibited in a dose-dependent manner by a myristoylated peptidefragment of the N-terminal region of MARCKS protein (the proposed siteof the protein's attachment to granule membranes). Accordingly, thismyristoylated peptide fragment of the N-terminal region of MARCKSprotein, as well as other active peptide fragments, are useful inmethods of inhibiting mucus secretion. As described further herein, theadministration of certain active fragments of MARCKS protein has beenfound to be capable of both increasing and decreasing mucus secretion byepithelial mucus-secreting cells.

The present inventors have discovered that antisense oligonucleotidesdirected against MARCKS protein block or inhibit mucin secretion, asdescribed further herein. Accordingly, such antisense oligonucleotidesfind use in methods of inhibiting mucus secretion.

Additionally, certain non-protein inhibitors of components in the mucussecretion signaling pathway illustrated in FIG. 4 inhibit mucussecretion in mucus-secreting cells, and are thus useful in the practiceof methods the present invention. For example, inhibitors of PKC such ascalphostin C, inhibitors of cyclic GMP such as Rp-8-Br-PET-cGMP,inhibitors of PKG such as Rp-8-Br-PET-cGMP, inhibitors of solubleguanylyl cyclase such as LY83585 and inhibitors of phosphatase such asokadaic acid each inhibit mucin secretion in cells stimulated by theabove-listed secretagogues. Accordingly, such inhibitors of componentsof the mucin secretion signaling pathway find use in methods ofinhibiting mucus secretion.

Other compounds that find use in the practice of the present inventionare those compounds that increase the amount (i.e., the concentration)of MARCKS protein in a mucus secreting cell. Such compounds have beenfound to increase mucus secretion in these cells. Although themechanisms of how these compounds increase the level or amount of MARCKSprotein in the cell are not known, possible mechanisms include (1)transcriptional modulation of the production of MARCKS mRNA (i.e.,through binding or alteration of transcription factors, or othermechanisms of modulating transcription) by these compounds, directly orindirectly, and (2) translational control or modulation of theexpression of MARCKS protein (e.g., by binding in the upstreamregulatory region of the MARCKS mRNA, thus potentially alteringsecondary structure that inhibits the amount of MARCKS proteinexpressed) by these compounds, either directly or indirectly. As usedherein, compounds that increase the amount of MARCKS protein in a mucussecreting cells may be proteins, peptides, or non-protein compounds. Inone embodiment of the invention, the compound is bacteriallipopolysaccharide (LPS). Preferably, the compound is a protein orpeptide. In a particularly preferred embodiment of the invention, thecompound is a cytokine. In a more particularly preferred embodiment ofthe invention, the compound that increases the amount of MARCKS proteinin a mucus secreting cell is the cytokine Tumor Necrosis Factor-alpha(TNF-α).

The present invention thus provides methods and compositions useful inregulating (increasing or decreasing) mucus secretion. Such methods andcompositions are useful in the treatment of medical conditions in whichmucus hypersecretion occurs, and are particularly useful in therespiratory tract. Methods and compositions of the present invention mayfurther be useful in treating medical conditions in which it is desiredto increase mucus secretion.

The methods and compositions of the present invention may be used toinhibit or reduce mucus secretion occurring from any mucus-secretingcell (such as goblet cells) or tissue (such as mucous membranes of theairways). While not wishing to be held to a particular theory, thepresent inventors also believe that the compounds and methods of thepresent invention may also be used to block the secretion ofinflammatory mediators from cells such as macrophages, neutrophils andmast cells. In this way, the present mucus-inhibitory compounds may havea dual function of decreasing mucus secretion and inflammation.

As discussed below, the present invention is also directed to activepeptide fragments of MARCKS protein that exhibit a bimodal effect whendelivered to mucus-secreting cells. At a certain dose level (amucus-enhancing amount), such peptides increase or enhance mucussecretion (compared to that which would occur in the absence of suchtreatment). At a different dose level, this enhancement is no longerobserved. An even more extreme dose results in the inhibition of ordecrease in mucus secretion (compared to that which would occur in theabsence of such treatment).

Accordingly, the present invention provides methods and compositions forregulating mucus secretion, by regulating the effects of MARCKS proteinin the mucus-secretory pathway. Such regulation can be achieved byadministering active fragments of MARCKS protein in pre-determinedamounts, administration of mucus-inhibiting amounts of MARCKS antisenseoligonucleotides, or administration of these or other compounds (aloneor in combination) that enhance or inhibit the MARCKS-related secretorypathway. Such compounds include those that block the dephosphorylatedMARCKS protein binding event that leads to mucin release. Such compoundsmay bind to and block the site that is bound by endogenous MARCKSprotein, or may bind to the MARCKS protein at the pertinent site. TheMANS peptide described herein is believed to compete with endogenousMARCKS protein for the pertinent binding site in the cell, thus blockingthe MARCKS-mediated release of mucin within the cell. Alternatively, anantibody directed to the N-terminal sequence of the MARCKS protein(e.g., the MANS sequence) would be predicted to bind to endogenousMARCKS protein and block binding.

While not wishing to be held to a single theory underlying the presentinvention, it is believed that compounds that increase MARCKS-relatedmucus secretion when administered to a mucus-secreting cell (such as theMA-PSD peptide; SEQ ID NO:2) may be binding to endogenous proteins inthe cell that would otherwise bind to MARCKS protein and inhibit MARCKSfrom completing a step in the mucus-secretion pathway. Calmodulin is onesuch endogenous inhibitor of MARCKS; calmodulin binds to MARCKS andprevents phosphorylation, thus preventing the MARCKS protein fromdisengaging from the plasma membrane. As used herein, “endogenousinhibitors of MARCKS protein” are compounds naturally present in a cellthat bind to MARCKS protein and prevent the completion of a step in theMARCKS-related mucus secretion pathway. A peptide or other compound thatbinds to a MARCKS inhibitor would leave more endogenous MARCKS proteinfree to function in the mucus secretion pathway. Thus, a method forincreasing mucus secretion is to administer to a mucus-secreting cell, acompound that binds to a MARCKS protein inhibitor.

It will be desirable, in many therapeutic situations, to maintain somelevel of mucus secretion (i.e., a basal or normal level), for theprotective effects of mucus. Maintenance of basal mucus secretion may beachieved by regulating the dose of the active compound utilized.Additionally, while not wishing to be held to a single theory of theinvention, the present inventors suggest that in some mucous membranes,a basal level of mucus secretion may be maintained by a pathway separatefrom the MARCKS-related pathway and stimulated mucus secretion.

The present invention provides methods and compositions able to decreaseor reduce mucus hypersecretion that occurs in many pathologicalconditions, including pathological conditions related to inflammatory,viral, bacterial, or genetic causes. In particular, the present methodsand compositions provide methods of treating airway diseases in whichmucus secretion is increased over that which occurs in the absence ofthe disease (i.e., is increased over basal levels, or overnormally-occurring levels of mucus secretion). Subjects to be treated bythe present methods include human and non-human subjects. Non-humansubjects include companion animals such as cats and dogs, as well aslivestock such as cattle, horses, sheep and swine.

The present methods and compositions may be used to reduce mucussecretion, or to inhibit mucus hypersecretion, in any secretoryepithelium, or epithelial cell, including but not limited to airwayepithelial cells (e.g., oral, nasal, bronchial), ocular epithelialcells, gastric or intestinal epithelial cells, and epithelial cellslining the reproductive tract (e.g., vaginal, cervical). As will beapparent to those skilled in the art based on the subject and thecondition being treated, it may be desirable to maintain a basal levelof mucus secretion, while reducing hypersecretion of mucus. As usedherein, a treatment that reduces or inhibits mucus secretion refers to atreatment that reduces the amount of secreted mucus compared to thatwhich would occur in the subject in the absence of such treatment.

The present invention also provides a method and compositions forincreasing or stimulating mucus secretion by epithelial cells, includingbut not limited to airway epithelial cells, ocular epithelial cells(corneal epithelium or conjunctiva), gastric or intestinal epithelialcells, and epithelial cells lining the reproductive tract. As usedherein, a treatment that increases, enhances or stimulates mucussecretion refers to a treatment that increases the amount of secretedmucus compared to that which would occur in the absence of suchtreatment. In particular, the present methods and compositions areuseful in increasing the secretion of mucus by ocular epithelial cells(to treat dry-eye conditions), and by vaginal epithelial cells (to treatvaginal dryness).

The peptides and compounds of the present invention block mucussecretion in response to known activators of PKC and protein kinase G(FIG. 1), and to physiologically relevant stimuli (e.g., UTP). (FIG. 2)

The present invention thus provides methods and compositions fortreating epithelial cells or epithelial tissue, where it is desirable todecrease the amount of mucus secreted by the cells or tissue. Thepresent invention also thus provides methods and compositions fortreating epithelial cells or epithelial tissue, where it is desirable toincrease the amount of mucus secreted by the cells or tissue. Inparticular the present invention provides methods and compositions fortreating respiratory conditions where it is desirable to decrease theamount of mucus present in the airways, or where it is desirable toincrease the amount of mucus present in the airways. Conditions suitablefor treatment by the present methods include human and animalinflammatory, viral or bacterial airway disease (e.g., asthma, chronicobstructive pulmonary disease, common cold, rhinitis, acute or chronicbronchitis, pneumonia, and kennel cough), allergic conditions (atopy,allergic inflammation), bronchiectasis, and certain genetic conditions(e.g., cystic fibrosis).

Mucus Secretion in the Airways

Normal mucus secretion in the lung plays an important role in clearinginhaled foreign particles and pathogens from the airways. Mucus trapsinhaled particles, and is then removed from the airways by ciliaryaction or by coughing. Above-normal levels of mucus secretion(hypersecretion) in the airways can lead to intraluminal mucusaccumulation, resulting in airflow obstruction and an increasedsusceptibility to infectious agents. Secretory cells in the airwaysinclude submucosal glands and superficial epithelial mucus cells (gobletcells).

Airway mucus secretion is an important determinant in the prognosis andclinical features of pulmonary diseases. Hypertrophy and/or hyperplasiaof airway secretory cells (bronchial glands and epithelial goblet cells)are often found in conditions associated with chronic airwayinflammation. In subjects with chronic bronchitis and bronchial asthma,goblet cell hyperplasia has been observed, with a two- to three-foldincrease in the numbers of goblet cells compared to controls. Cutz etal., Histopathology 2:407-421 (1978), Glynn & Michaels Thorax 15:142-153(1960). Inflammation of the airways may induce mucus hypersecretion bymultiple mechanisms, including the release of chemical mediators fromsurrounding tissues and cells. Airway mucus hypersecretion is aparticularly dominant clinical finding in cystic fibrosis, bronchitis,chronic obstructive pulmonary disease (COPD), emphysema, and bronchialasthma. See, e.g., Airway Secretion, Takishima and Shimura (Eds.),Marcel Dekker Inc., 1994. The presence of excessive bronchial mucus canlead to respiratory failure and bacterial infection. Lungs of asthmaticpatients, at autopsy, often show the presence of excessive bronchialmucus and mucus plugging. Methods of reducing airway mucus secretionwould be useful for the treatment of such conditions, as well as intreating bacterial or viral infections (e.g., pneumonia, influenza, andthe common cold); in animals, such methods are further useful intreating kennel cough and equine COPD.

Various methods are currently in use to reduce mucus secretion whenneeded in disease states. Some therapies act to decrease the signals orstimuli that upregulate mucus secretion. For example, inflammatorymediators may upregulate mucus secretion; steroid treatments are oftenused to decrease inflammation and thus indirectly decrease mucussecretion. Antihistamines are used to block the responses to allergenswhich can trigger attacks of allergic asthma. The thickened mucuspresent in patients with cystic fibrosis is removed by compressiontherapy, and infections occurring due to the thickened mucus are treatedwith antibiotics. The methods and compounds of the present inventionvary from the above treatments in that cellular secretion of mucus inresponse to a variety of stimuli is directly blocked at the cellularlevel.

Ocular Mucus Layer

The mucus layer on the ocular surface is important in maintaining andspreading the tear film, and is required for normal functioning of theeyes. The ocular surface epithelia has been shown to express multiplemucin genes. Gipson, Adv. Exp. Med. Biol. 438:221 (1998). Variousdiseases and syndromes result in pathological “dry-eye” conditions,including keratoconjunctivitis sicca, Stevens-Johnson syndrome, ocularpemphigoid, and surgery- or radiation-induced dry-eye. The ocularsurface epithelia in such diseases undergoes changes, which may includeloss of goblet cells, mucin deficiency, and keratinization. Lower gobletcell densities in the ocular epithelia have been demonstrated in thesesyndromes. Ralph, Invest. Opthalmol. 14:299 (1975).

In addition to subjects in which dry-eye causes discomfort in dailylife, many individuals have “marginal” dry-eye which may only presentdifficulties when the subject attempts to wear contact lenses. Contactlens intolerance is frequently due to insufficient tear film. Jurkus etal., J. Am. Optom. Assoc. 65:756 (1994); Toda et al., Br. J. Ophthal.80:604 (1996). Existing treatments for dry-eye include topical use oftretinoin (Tseng, J. Am. Acad. Dermatol. 15:860 (1986)) or retinoic acid(Driot & Bonne, Invest. Ophthalmol. 33:190 (1992)).

Increasing mucus secretion in the eyes is desirable where a lack ofocular mucus affects the normal function of the eye. Additionally,increasing mucus secretion in the eye is desirable as an aid in wearingcontact lenses.

Administration

The method of the present invention can be used to reduce (i.e.,decrease or inhibit) or to enhance (i.e., increase or stimulate) theproduction of mucus secretions by mucous membranes or mucus-secretingcells, in a subject in need of such treatment for any reason. Usingmethods of administration as are known in the art, the present therapiescan be directed to the mucous membranes or mucus-secreting cells of aparticular target organ (including but not limited to the oral cavity,nasal cavity, lungs, gastrointestinal tract, eye and reproductivetract), in order to reduce or increase the amount of mucus secreted by,or retained upon, the surfaces being treated. The change (reduction orincrease) in mucus is assessed by comparison to that which was presentprior to treatment (or in the absence of treatment), or to that whichwould be expected in the absence of such treatment in view of thesubject's condition.

The methods of the present invention may be used in conjunction withother therapies or compounds, including steps to remove retained mucussecretions from the airways of subjects prior to the step ofadministering the present compounds. This facilitates application of theactive agent to the respiratory epithelia during the administering step.Such removal of retained mucus secretions can be carried out by anysuitable physical or medicinal means as are known in the art.

Mucosal delivery of peptide-based drugs is discussed in Chien, NovelDrug Delivery Systems, Chapter 4 (Marcel Dekker, 1992); nasal drugdelivery is discussed in Chien, supra, in Chapter 5. See also Chang etal., Nasal Drug Delivery, “Treatise on controlled Drug Delivery”,Chapter 9 (Marcel Dekker, 1992). Agents known to enhance absorption ofdrugs through the skin are described in Sloan, Chapter 5, “Prodrugs:Topical and Ocular Drug Delivery” (Marcel Dekker, 1992). The peptides ofthe present invention can be administered into target cells directly,for example using liposomes. It is expected that those skilled in theart may adapt such techniques and other known drug delivery techniquesfor use with the compounds of the present invention without undueexperimentation.

Pharmaceutical compositions for use in the present method of treatmentinclude those suitable for inhalation, oral, rectal, vaginal, topical(including buccal, dermal and ocular) administration. The compositionsmay be prepared by any of the methods well known in the art. The mostsuitable route of administration in any case will depend upon thelocation of the tissue to be treated, the nature and severity of thecondition being treated, and the particular active compound which isbeing used, as will be apparent to those skilled in the art. The dosageof active compound for treatment of diseases of the respiratory tractwill vary depending on the condition being treated and the state of thesubject. One skilled in the art would be able to determine appropriatedosages of specific compounds without undue experimentation, using doseresponse studies as are known in the art.

The active compounds disclosed herein may be administered to the airwaysof a subject by any suitable means, but are preferably administered bygenerating an aerosol comprised of respirable particles, the respirableparticles comprised of the active compound, which particles the subjectinhales. The respirable particles may be liquid or solid. The particlesmay optionally contain other therapeutic ingredients. (See, e.g., U.S.Pat. No. 5,849,706 to Molina y Vedia et al.)

In methods of treating the bronchi and/or alveoli, particles comprisedof active compound for practicing the present invention should includeparticles of respirable size: that is, particles of a size sufficientlysmall to pass through the mouth and larynx upon inhalation and into thebronchi and alveoli of the lungs. In general, particles ranging fromabout 0.5 to 10 microns in size (more particularly, less than about 5microns in size) are respirable. Particles of non-respirable size whichare included in the aerosol tend to deposit in the throat and beswallowed, and the quantity of non-respirable particles in aerosolsintended for treatment of the alveoli and/or bronchi is preferablyminimized. For nasal administration, a particle size in the range of10-500 microns is preferred to ensure retention in the nasal cavity.

Liquid pharmaceutical compositions of active compound for producing anaerosol can be prepared by combining the active compound with a suitablevehicle, such as sterile pyrogen free water.

Administration of the active compounds may be carried outtherapeutically or prophylactically (e.g., before substantial lungblockage due to retained mucus secretions has occurred, or at a timewhen such retained secretions have been at least in part removed, asdiscussed above.)

Aerosols of liquid particles comprising the active compound may beproduced by any suitable means, such as with a nebulizer. See, e.g.,U.S. Pat. No. 4,501,729. Nebulizers are commercially available deviceswhich transform solutions or suspensions of the active ingredient into atherapeutic aerosol mist either by means of acceleration of a compressedgas, typically air or oxygen, through a narrow venturi orifice or bymeans of ultrasonic agitation. Suitable formulations for use innebulizers consist of the active ingredient in a liquid carrier,typically water or a dilute aqueous alcoholic solution, and preferablymade isotonic with body fluids.

Aerosols of solid particles comprising the active compound may likewisebe produced with any solid particulate medicament aerosol generator. Oneillustrative type of solid particulate aerosol generator is aninsufflator.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). Formulations for oral administration may optionallyinclude enteric coatings known in the art to prevent degradation of theformulation in the stomach and provide release of the drug in the smallintestine.

Formulations suitable for rectal or vaginal administration may bepresented as unit dose suppositories. These may be prepared by admixingthe active compound with one or more conventional solid carriers, forexample, cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the eye, mouth, nasalor other surfaces may take the form of an ointment, cream, lotion,paste, gel, spray, aerosol, or oil.

Peptides

The myristoylated alanine-rich C kinase substrate (MARCKS) protein is amajor cellular substrate for protein kinase C. MARCKS is regulated in acell-, tissue- and developmental stage-specific manner, and expressionof MARCKS can be stimulated by various cytokines. MARCKS has beenidentified in human, bovine, rodent and avian species. Harlan et al., J.Biol. Chem. 266:14399 (1991); Graff et al., J. Biol. Chem. 266:14390(1991); Graff et al., Mol. Endocrinol. 3:1903 (1989); Stumpo et al.,Proc. Natl. Acad. Sci. USA 86:4012-16 (June 1989).

Peptides corresponding to the MARCKS protein are commercially available.A MARCKS “psd peptide” is available from BIOMOL (Plymouth Meeting, Pa.),having the sequence KKKKKRFSFK KSFKLSGFSF KKNKK (SEQ ID NO:2). See P.Blackshear, J. Biol. Chem. 268:1501 (1993).

The present inventors have identified two specific active fragments ofMARCKS protein that are able to affect mucus secretion. A myristoylatedpolypeptide, 24 amino acids in length, with sequence Myristicacid-GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), is referred to herein asthe MANS peptide for myristolated N-terminal sequence. The peptideinhibits secretion of mucus from mucous membranes and mucus-secretingcells, including human airway epithelial cells. The present inventors'data suggests that this peptide blocks the attachment of MARCKS proteinto the mucin granule, thus blocking or inhibiting the release of mucingranules and the secretion of mucus by the cell.

A second peptide corresponding to the PSD (phosphorylation) site ofMARCKS was also tested. At some concentrations this peptide stimulatesmucus secretion, while at other doses (higher) it has no effect onstimulated secretion (FIG. 3), and it is predicted that even higherdoses will decrease stimulated mucus secretion. The PSD peptide sequenceis myristic acid—KKKKKRFSFKKSFKLSGFSFKKNKK (SEQ ID NO:2), referred toherein as the MA-PSD peptide. While not wishing to be held to a singletheory underlying the present invention, the inventors believe thatMARCKS protein fragments that are able to increase mucus secretion (suchas the MA-PSD peptide, SEQ ID NO:2) may be binding to endogenousproteins in the cell that competitively inhibit the phosphorylation ofMARCKS, thus inhibiting the release of MARCKS from the plasma membraneinto the cell interior (see FIG. 4). One such inhibitor of MARCKSphosphorylation is calmodulin. Other “MARCKS inhibitors”, for purposesof the present invention, are those endogenous compounds that preventthe MARCKS protein from completing a necessary step in themucus-secretion pathway. MARCKS inhibitors may thus act to inhibit thephosphorylation or the dephosphorylation of MARCKS (each of which isnecessary in the present pathway), or bind to MARCKS to prevent itsbinding to the mucin granule membrane. Compounds of the presentinvention that increase the secretion of mucus may be acting by bindingto such endogenous inhibitors, thus freeing endogenous MARCKS protein tocomplete the mucus-secretion pathway.

Thus, peptide fragments of the MARCKS protein may be designed, testedand selected for their ability to inhibit or enhance mucus secretion,using the present disclosure and methods known in the art.

The nucleotide and amino acid sequences of human MARCKS cDNA and proteinas reported by Harlan et al., J. Biol. Chem. 266:14399 (1991) (GenBankAccession No. M68956) are provided as SEQ ID NO:3 and SEQ ID NO:4. Thenucleotide and amino acid sequences of human MARCKS cDNA and protein asreported by Sakai et al., Genomics 14:175 (1992) are provided as SEQ IDNO:5 and SEQ ID NO:6. An additional publication (Harlan et al., J. Biol.Chem. 266(22):14399 (1991) provides a nucleotide sequence for humanMARCKS that differs from that of Sakai et al. at nucleotides 619 and724; in this sequence, G is substituted for T at position 619 and C issubstituted for G at position 724. Additional allelic variants of humanand other MARCKS proteins would be expected.

While not wishing to be held to a single theory underlying the presentinvention, the present inventors propose that the pathway for theinvolvement of MARCKS in mucus secretion in airway epithelium is asshown in FIG. 4. It is currently believed that active peptide fragmentsof MARCKS affect mucus secretion at the level of the interaction ofMARCKS with the mucin granules, which contain the major proteincomponents of mucus. As shown in FIG. 4, the present inventors believethat MARCKS must be dephosphorylated to bind to the mucin granule, whichtriggers mucin exocytosis and results in mucus secretion.

The methods of the present invention include the use of isolated DNAmolecules encoding the peptides of the present invention. Such isolatedDNA molecules are useful in producing the therapeutic peptides, and mayadditionally be used in an appropriate gene expression vector for genetherapy, using methods as are known in the art for the expression of thepeptide in vivo. Cell-specific or inducible promoters may further beused to control the expression of the therapeutic peptide in vivo.Methods of delivering DNA encoding a desired peptide to achieve atherapeutic effect is disclosed, e.g. in U.S. Pat. Nos. 5,580,859 and5,703,055 to Felgner et al.

Analogs of the therapeutic peptides disclosed herein are an aspect ofthe present invention. As used herein, an “analog” is a chemicalcompound similar in structure to a first compound, and having a similarphysiologic action as the first compound. With particular reference tothe present invention, MARCKS peptide analogs are those compounds which,while not having the exact amino acid sequences of the native MARCKSfragment, are capable of binding to the same sites as the native MARCKSfragment. Such analogs may be peptide or non-peptide analogs, includingnucleic acid analogs, as described in further detail below.

In protein molecules which interact with a receptor, the interactionbetween the protein and the receptor must take place atsurface-accessible sites in a stable three-dimensional molecule. Byarranging the critical binding site residues in an appropriateconformation, peptides which mimic the essential surface features of thep20 ligands may be designed and synthesized in accordance with knowntechniques.

Methods for determining peptide three-dimensional structure and analogsthereto are known, and are sometimes referred to as “rational drugdesign techniques”. See, e.g., U.S. Pat. No. 4,833,092 to Geysen; U.S.Pat. No. 4,859,765 to Nestor; U.S. Pat. No. 4,853,871 to Pantoliano;U.S. Pat. No. 4,863,857 to Blalock; (applicants specifically intend thatthe disclosures of all U.S. Patent references cited herein beincorporated by reference herein in their entirety). See also Waldrop,Science, 247, 28029 (1990); Rossmann, Nature, 333, 392-393 (1988); Weiset al., Nature, 333, 426-431 (1988); James et al., Science, 260, 1937(1993) (development of benzodiazepine peptidomimetic compounds based onthe structure and function of tetrapeptide ligands).

In general, those skilled in the art will appreciate that minordeletions or substitutions may be made to the amino acid sequences ofpeptides of the present invention without unduly adversely affecting theactivity thereof. Thus, peptides containing such deletions orsubstitutions are a further aspect of the present invention. In peptidescontaining substitutions or replacements of amino acids, one or moreamino acids of a peptide sequence may be replaced by one or more otheramino acids wherein such replacement does not affect the function ofthat sequence. Such changes can be guided by known similarities betweenamino acids in physical features such as charge density,hydrophobicity/hydrophilicity, size and configuration, so that aminoacids are substituted with other amino acids having essentially the samefunctional properties. For example: Ala may be replaced with Val or Ser;Val may be replaced with Ala, Leu, Met, or Tie, preferably Ala or Leu;Leu may be replaced with Ala, Val or Ile, preferably Val or Ile; Gly maybe replaced with Pro or Cys, preferably Pro; Pro may be replaced withGly, Cys, Ser, or Met, preferably Gly, Cys, or Ser; Cys may be replacedwith Gly, Pro, Ser, or Met, preferably Pro or Met; Met may be replacedwith Pro or Cys, preferably Cys; His may be replaced with Phe or Gln,preferably Phe; Phe may be replaced with His, Tyr, or Trp, preferablyHis or Tyr; Tyr may be replaced with His, Phe or Trp, preferably Phe orTrp; Trp may be replaced with Phe or Tyr, preferably Tyr; Asn may bereplaced with Gln or Ser, preferably Gln; Gln may be replaced with His,Lys, Glu, Asn, or Ser, preferably Asn or Ser; Ser may be replaced withGln, Thr, Pro, Cys or Ala; Thr may be replaced with Gln or Ser,preferably Ser; Lys may be replaced with Gln or Arg; Arg may be replacedwith Lys, Asp or Glu, preferably Lys or Asp; Asp may be replaced withLys, Arg, or Glu, preferably Arg or Glu; and Glu may be replaced withArg or Asp, preferably Asp. Once made, changes can be routinely screenedto determine their effects on function with enzymes.

Non-peptide mimetics of the peptides of the present invention are alsoan aspect of this invention. Non-protein drug design may be carried outusing computer graphic modeling to design non-peptide, organic moleculeswhich bind to sites bound by the native MARCKS fragments disclosedherein. See, e.g., Knight, BIO/Technology, 8, 105 (1990). Itzstein etal, Nature, 363, 418 (1993); Lam et al, Science, 263, 380 (January 1994)(rational design of bioavailable nonpeptide cyclic ureas that functionas HIV protease inhibitors). Analogs may also be developed by generatinga library of molecules, selecting for those molecules which act asligands for a specified target, and identifying and amplifying theselected ligands. See, e.g., Kohl et al., Science, 260, 1934 (1993).Techniques for constructing and screening combinatorial libraries ofoligomeric biomolecules to identify those that specifically bind to agiven receptor protein are known. Suitable oligomers include peptides,oligonucleotides, carbohydrates, non-oligonucleotides (e.g.,phosphorothioate oligonucleotides; see Chein. and Engineering News, page20, 7 Feb. 1994) and nonpeptide polymers (see, e.g., “peptoids” of Simonet al., Proc. Natl. Acad. Sd. USA, 89, 9367 (1992)). See also U.S. Pat.No. 5,270,170 to Schatz; Scott and Smith, Science, 249, 386-390 (1990);Devlin et al., Science 249, 404-406 (1990); Edgington, BIO/Technology,11, 285 (1993). Peptide libraries may be synthesized on solid supports,or expressed on the surface of bacteriophage viruses (phage displaylibraries). Known screening methods may be used by those skilled in theart to screen combinatorial libraries to identify suitable peptideanalogs. Techniques are known in the art for screening synthesizedmolecules to select those with the desired activity, and for labellingthe members of the library so that selected active molecules may beidentified. See, e.g., Brenner and Lerner, Proc. Natl. Acad. Sci. USA,89, 5381 (1992); PCT U593/06948 to Berger et al.; Simon et al., Proc.Natl. Acad. Sci. USA, 89, 9367, (1992); U.S. Pat. No. 5,283,173 toFields et al.

As used herein, “combinatorial library” refers to collections of diverseoligomeric biomolecules of differing sequence, which can be screenedsimultaneously for activity as a ligand for a particular target.Combinatorial libraries may also be referred to as “shape libraries”,i.e., a population of randomized polymers which are potential ligands.The shape of a molecule refers to those features of a molecule thatgovern its interactions with other molecules, including Van der Waals,hydrophobic, electrostatic and dynamic.

Nucleic acid molecules may also act as ligands for receptor proteins.See, e.g., Edgington, BIO/Technology, 11, 285 (1993). U.S. Pat. No.5,270,163 to Gold and Tuerk describes a method for identifying nucleicacid ligands for a given target molecule by selecting, from a library ofRNA molecules with randomized sequences, those molecules that bindspecifically to the target molecule. A method for the in vitro selectionof RNA molecules immunologically cross-reactive with a specific peptideis disclosed in Tsai, Kenan and Keene, Proc. Natl. Acad. Sci. USA, 89,8864 (1992) and Tsai and Keene, J. Immunology, 150, 1137 (1993).

Antisense Oligonucleotides

The present inventors have further demonstrated that antisenseoligonucleotides directed against MARCKS mRNA decreases (inhibits) mucussecretion in human airway epithelial cells. (See Example 6 and FIG. 5).

It has been demonstrated that antisense oligonucleotides that arecomplementary to specific RNAs can inhibit the expression of cellulargenes as proteins. See Erickson and Izant, Gene Regulation: Biology OfAntisense RNA And DNA, Vol. 1, Raven Press, New York, 1992. For example,selective inhibition of a p21 gene that differed from a normal gene by asingle nucleotide has been reported. Chang et al., Biochemistry 1991,30:8283-8286. Many hypotheses have been proposed to explain themechanisms by which antisense oligonucleotides inhibit gene expression,however, the specific mechanism involved may depend on the cell typestudied, the RNA targeted, the specific site on the RNA targeted, andthe chemical nature of the oligonucleotide. Chiang et al., J. Biol.Chem. 1991, 266:18162-18171; Stein and Cohen, Cancer Res. 1988,48:2659-2668.

The present invention provides oligonucleotides substantiallycomplementary to a MARCKS protein nucleotide sequence that occursendogenously in a mucus-secreting cell. Such oligonucleotides are usefulin decreasing mucus production by cells into which they are delivered.“Nucleotide sequence” refers to a polynucleotide formed from a series ofjoined nucleotide units. The term “substantially complementary”, as usedherein, refers to that amount of sequence complementarity between theoligonucleotide and a MARCKS gene nucleotide sequence which allows forinterstrand hybridization under physiological conditions and enables theoligonucleotide to inhibit the expression of the MARCKS gene.Interstrand hybridization is the interaction between the oligonucleotideand the MARCKS nucleotide sequence. The potential of forming a stableinterstrand hybrid can be determined by those skilled in the art usingmethods known in the art, such as, for example, determination of themelting temperature for the hybrid by mathematical modeling or empiricalanalysis, or solid support nucleic acid hybridizations. (See, e.g.,Marmur and Doty, J. Mol. Biol. 1962, 5, 113).

Antisense DNAs used in the present invention are able to produce thecorresponding antisense RNAs. An antisense RNA molecule has thenucleotide bases in the reverse or opposite order for expression. Suchantisense RNAs are well known in the art, see e.g., U.S. Pat. No.4,801,540 to Calgene Inc.

As used herein, the term “MARCKS nucleotide sequence” refers to anynucleotide sequence derived from a gene encoding a MARCKS protein,including, for example, DNA or RNA sequence, DNA sequence of the gene,any transcribed RNA sequence, RNA sequence of the pre-mRNA or mRNAtranscript, and DNA or RNA bound to protein.

Oligonucleotides targeted to sequences in MARCKS genes can be used toinhibit mucus production in epithelial cells. The oligonucleotide may beany length of sequence capable of forming a stable hybrid with theendogenous MARCKS nucleotide sequence under physiologic conditions. Itis preferred that the length of the oligonucleotide be between 5 and 200nucleotides. It is more preferred that the oligonucleotide be between 10and 50 nucleotides in length. It is most preferred that theoligonucleotide be between 15 and 25 nucleotides in length.

The nucleotides of the oligonucleotides may be any known in the artincluding natural and synthetic moieties. The term “oligonucleotide” asused herein refers to a polynucleotide formed from joined nucleotides.Moreover, the term “oligonucleotide” includes naturally occurringoligonucleotides or synthetic oligonucleotides formed from naturallyoccurring subunits or analogous subunits designed to confer specialproperties on the oligonucleotide so that it is more stable inbiological systems or binds more tightly to target sequences. It alsoincludes modifications of the oligonucleotides such as chemicallylinking them to other compounds that will enhance delivery to cells orto the nucleus and other compartments of cells. Oligonucleotides of theinvention may be synthesized by any method known in the art, includingsynthetic chemical methods. See, e.g., Vu and Hirschbein, TetrahedronLett. 1991, 32:30005-30008. Oligonucleotides may be modified viachemical methods known to those skilled in the art, includingencapsulation in liposomes, or chemical linkage to steroids, antibodies,and cell receptors.

A preferred embodiment of the invention is an oligonucleotidecomplementary to an endogenous MARCKS nucleotide sequence found in thecell to be treated, or having sufficient complementarity to allow stableinterstrand hybridization between the oligonucleotide and an endogenousMARCKS nucleotide, and that inhibits the expression of the MARCKS gene.A preferred oligonucleotide is one that is complementary to a MARCKSnucleotide sequence derived or selected from a mammal, in particular, ahuman.

The oligonucleotides of the present invention may beoligodeoxyribonucleotides or oligoribonucleotides, including modifiedoligodeoxynucleotides and oligoribonucleotides. Moreover, theoligonucleotides of the invention may be comprised of combinations ofdeoxyribonucleotides and ribonucleotides. Further, oligonucleotides ofthe invention may also include modified subunits. For example, theinvention may include phosphorothioate oligodeoxyribonucleotides. It ispreferred that the oligonucleotides of the invention be modified toincrease stability and prevent intracellular and extracellulardegradation. It is more preferred that the oligonucleotides of theinvention be modified to increase their affinity for target sequences,and their transport to the appropriate cells and cell compartments whenthey are delivered into a mammal in a pharmaceutically active form.

It is preferred that the oligonucleotides of the invention be antisenseoligonucleotides. The oligonucleotides of the invention may be targetedto a non-coding portion of a MARCKS or targeted to coding sequences ofthe gene, and may include an intron-exon junction (i.e., severalnucleotides on either or both sides of the intron-exon junction).

The oligonucleotides of the invention may be administered by any methodthat produces contact of the oligonucleotide with the target tissue orcell in the subject being treated, including but not limited to oraladministration, topical administration, and inhalation. Thepharmaceutical compositions comprising the oligonucleotides may be insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. The dosageadministered varies depending upon factors such as: pharmacodynamiccharacteristics; its mode and route of administration; age, health, andweight of the recipient; nature and extent of symptoms; kind ofconcurrent treatment; and frequency of treatment and the effect desired.Effective dosages are those which are able to inhibit mucus productionin the airways at a level which alleviates, reduces, or eliminates thesymptoms or conditions associated with the mucus production.

The oligonucleotides may be administered singly, or in combination withother compounds of the invention, other pharmaceutical compounds, ortherapies. The oligonucleotides are preferably administered with apharmaceutically acceptable carrier or diluent selected on the basis ofthe selected route of administration and standard pharmaceuticalpractice.

Inhibition of secretion of mucus, via inhibition of MARCKS proteinfunction in epithelial secretory cells, is a focus of this invention. Toachieve this end, the invention provides methods of inhibiting mucussecretion which comprises contacting a mucus-secretory cell with aMARCKS gene expression inhibitory amount of an oligonucleotidesubstantially complementary to an endogenous MARCKS gene nucleotidesequence. The invention also includes a method whereby the contactingstep comprises lipofectin as a carrier for the oligonucleotide. Theoligonucleotides of the invention are administered to mammals or avians,and preferably to humans, in therapeutically effective amounts orconcentrations which are effective to inhibit or reduce mucus productionin the target tissue or organ.

The oligonucleotides of the invention will be capable of reaching theirintracellular target to inhibit or reduce the expression of MARCKSprotein therein. The invention therefore provides methods of inhibitingmucus secretion which comprise contacting at least one element of MARCKSgene expression machinery with a gene expression inhibitory amount of anoligonucleotide. For the purposes of the invention, the elements of thegene expression machinery may comprise any nucleotide sequence of aMARCKS gene, the nucleotide sequence of spliced mRNAs transcribed from agene, unspliced RNAs and partially spliced RNAs transcribed from a gene,DNA-RNA hybrids comprising sequence derived from a gene, such as inactively transcribing genes, RNA transcribed from a gene bound toprotein, and any molecule or structure known in the art to be involvedin gene expression.

U.S. Pat. No. 5,858,784 to Debs et al. provides a method ofadministering nucleic acids to the lung cells of a subject by preparinga liposome-nucleic acid mixture suitable for nebulization, nebulizingthe mixture, and depositing the resulting nebulized mixture in the lungsof the subject. The nucleic acid sequence may include DNA sequenceswhich encode polypeptides which are directly or indirectly responsiblefor a therapeutic effect, or active nucleotide sequences such asantisense sequences and ribozymes. The nucleic acid constructs can beprovided to the cells of the subject as expression cassettes;preferably, the construct does not become integrated into the host cellgenome and is introduced into the host as part of a non-integratingexpression vector. (The disclosures of all US patents cited herein areintended to be incorporated herein in their entirety.)

Double Stranded RNA and Ribozymes

It has recently been shown that the introduction of exogenousdouble-stranded RNA (dsRNA) can specifically disrupt the activity ofgenes containing homologous sequences, possibly by post-transcriptionaleffects. Montgomery et al., Proc. Natl. Acad. Sci. USA 95:15502 (1998);Ngo et al., Proc. Natl. Acad. Sci. USA 95:14687 (1998). Accordingly, themethods of the present invention may be carried out by introducingexogenous dsRNA into a mucus-secreting cell, where the dsRNA hassufficient sequence similarity to the RNA of an endogenous MARCKS geneto result in a reduction in MARCKS protein in the cell (compared to thatwhich would occur in the absence of the exogenous dsRNA).

The administration of dsRNA may be carried out using the methodsdiscussed above regarding peptide and antisense oligonucleotideadministration.

In an alternate embodiment of the present invention, DNA encoding anenzymatic RNA molecule (ribozyme) may be introduced into the targetcell. Ribozymes are directed against and cleave the mRNA transcript ofthe cell's endogenous MARCKS protein. DNA encoding enzymatic RNAmolecules may be produced in accordance with known techniques (see e.g.,U.S. Pat. No. 4,987,071. Production of such an enzymatic RNA moleculeand disruption of MARCKS protein production affects mucus production bythe target cell in essentially the same manner as production of anantisense RNA molecule.

Methods of Screening

The present invention also provides a method of screening compounds fortheir ability to affect (enhance or inhibit) mucus production.Combinatorial chemistry processes as are known in the art may be used togenerate large numbers of structurally diverse compounds, which can thenbe screened. Such screening methods comprise providing a culture ofmucus-secreting cells, such as the cultures of normal human bronchialepithelial cells described in Example 1 herein. A test compound isadministered to the cells, and the cells may also be exposed to acompound known to stimulate mucus production (e.g., PMA, UTP,8-bromo-cGMP). The test compound and the stimulatory compound may beadministered to the cells, for example, by exposing the cells to mediacontaining the compounds. The cells may, for example, be pre-incubatedwith the test compound first, then co-incubated with the stimulatorycompound and the test compound. Alternatively, the pre-incubation stepmay be omitted. The ability of the test compound to bind to either themucin granule membrane (or a mucin granule membrane-related receptor) orto endogenous MARCKS protein at the mucin granule membrane binding siteis assessed by detecting whether the test compound inhibits binding ofendogenous MARCKS to the mucin granule. Such detection can be carriedout using methods known in the art, for example, by labelling the testcompound with a detectable molecule.

Molecules detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical and optical means are known. Opticallydetectable molecules include fluorescent labels (fluorescein, Texas Red,Green Fluorescent Protein). Methods for viewing intact cells are known,including real-time confocal laser-scanning microscopy and two-photonlaser-scanning microscopy.

Mucus secreted by the cells may also be measured after a pre-determinedtime period, for example using an ELISA assay as is known in the art.The mucus secretion of the cells exposed to the test compound can alsobe compared to that of control cells that were not exposed to the testcompound. A decrease in mucus secretion by the test cells compared tothe control cells indicates that the test compound inhibits mucussecretion, and an increase in mucus secretion by the test cells comparedto the control cells indicates that the test compound enhances mucussecretion.

The following examples are provided to more fully illustrate the presentinvention and should not be construed as limiting thereof.

Example 1 In Vitro Assessment of Mucus Secretion

Cell Culture System: Expansion and cryopreservation. Primary normalhuman bronchial epithelial (NHBE) cells (Clonetics, San Diego, Calif.)were seeded into vented T75 tissue culture flasks (500 cells/cm²) inbronchial epithelial basal medium (BEBM; Clonetics, San Diego, Calif.)containing 25 ng/ml human recombinant epidermal growth factor (EGF;Intergen, Purchase, N.Y.), 65 ng/ml bovine pituitary extract (preparedby the methods of Bertolero et al. Exp Cell Res 155:64, 1984), 5×10⁻⁸Mall-trans retinoic acid, 1.5 μg/ml bovine serum albumin (Intergen,Purchase, N.Y.), 20 IU/ml nystatin (Gibco, Grand Island, N.Y.), 0.5μg/ml hydrocortisone, 5 μg/ml insulin, 10 μg/ml transferrin, 0.5 μg/mlepinephrine, 6.5 ng/ml triiodothyronine, 50 μg/ml gentamicin, and 50μg/ml amphotericin-B (Clonetics; San Diego, Calif.). Once confluent,cultures were dissociated with trypsin/EDTA and frozen as passage-2according to the methods of Clonetics Corporation.

Air-liquid interface culture of NHBE cells. Following the expansion,NHBE cells were cultured in air/liquid interface according to themethods of Gray and co-workers with minor modifications (Gray et al. AmJ Respir Cell Mol Biol 14:104, 1996). The air-liquid interface culturewas initiated by seeding NHBE cells (passage-2, 2×10⁴ cells/cm²) onTranswell-clear culture inserts (24.5 mm, 0.45 μm pore size; Costar,Cambridge, Mass.) that were thin coated with rat tail collagen, type I(Collaborative Research, Bedford, Mass.). Cells were cultured submergedto 70% confluency (5-7 days) in a 1:1 mixture of bronchial epithelialcell growth medium (Clonetics, San Diego, Calif.):Dulbecco's modifiedEagles medium with high glucose (BEGM:DMEM-H), containing the samesupplements as described above with the exception of EGF (0.5 ng/ml).When cultures were 70% confluent, the air-liquid interface was createdby removing the apical medium, and the basal medium (BEGM:DMEM-H) waschanged daily thereafter. Cells were then cultured for an additional 14days in air-liquid interface, a total of 21 days in culture.

Mucus ELISA: Mucus secreted from the airway epithelial cells in vitroafter stimulation by activators was assessed using an ELISA (antibodycapture method) (E. Harlow, D. Lane. “Antibodies: A Laboratory Manual.”New York: Cold Spring Harbor Laboratory Press, 1988), wherein thecollected mucus is bound directly to the ELISA plate. Mucus was detectedusing an antibody raised against monkey airway mucus (Lin et al. Am JRespir Cell Mol Biol 1:41, 1989).

Example 2 MARCKS mRNA in Human Bronchial Epithelial Cells

MARCKS messenger RNA was detected in human bronchial epithelial cellsgrown in air/liquid interface culture by Northern analysis (Ausubel etal., eds. “Current Protocols in Molecular Biology.” New York: John Wiley& Sons, 1992) using a human MARCKS cDNA (approximate length 1 kb) as aradiolabelled probe. MARCKS message increases as these cells become moredifferentiated when maintained in an air/liquid interface culture.

To detect MARCKS protein in these cells, cells were labeled with³H-myristic acid (as MARCKS is myristoylated) for 16 hours in media.Cells were lysed, and MARCKS protein was immunoprecipitated according tothe method of Spizz & Blackshear (J Biol Chem 271:553, 1996) usingmonoclonal antibody 2F12 (a gift from the Blackshear laboratory).

MARCKS within the airway epithelial cells was found to be phosphorylatedby the PKC activator, PMA (100 nM), while 4α-PMA (a phorbol estercontrol which does not activate PKC), did not phosphorylate MARCKS.Phosphorylation of MARCKS by PMA was attenuated by Calphostin C (500nM). NHBE cells also contained substantial amounts of cGMP-dependentprotein kinase type 1α. (PKG-1α) activity, which was localized to thecytosolic fraction. The cells exhibited constitutive PKG activity whichwas increased by incubation with 100 μM dibutyryl cGMP. In addition, thephosphorylation of MARCKS induced by PMA was reversed by incubation with8-Br-cGMP (10 μM). Okadaic acid (500 nM) inhibited this effect. Theseresults indicate that 8-Br-cGMP activates a phosphatase (type 1 or 2A),which dephosphorylates MARCKS.

Example 3 Blocking of Mucin Secretion by Peptide MANS

The effect on mucus secretion of a myristoylated peptide containing thefirst 24 amino acids of the human MARCKS protein (MANS; myristoylatedN-terminal sequence; SEQ ID NO:1) was tested. Cultured normal humanbronchial epithelial cells as described above were used. Test cells wereco-incubated for 15 minutes in apical and basolateral media containing1, 10 or 100 μM of MANS peptide, and then co-incubated for 15 minuteswith the peptide and 100 nM PMA plus 1 μM 8-Br-cGMP (columns 3-5 of FIG.1A). Control cells were not exposed to MANS peptide but werepreincubated in media only (column 1 of FIG. 1A) or media containing PMAand 8-Br-cGMP (column 2 of FIG. 1A). Single asterisks (*) indicate thatthe response was statistically different than the media control (column1), and double asterisks (**) indicate that the response wasstatistically different than that observed in stimulated cells that werenot exposed to the MANS peptide (column 2).

Stimulation by PMA and 8-Br-cGMP caused at least a 100% increase inmucus secretion over control levels. This increase, however, was blockedby pre- and co-incubation with the MANS peptide. Levels of secretedmucus fell to control values when 10 μM peptide was used, and levels ofsecreted mucus were well below control values following incubation with100 μM MANS peptide (FIG. 1A).

The MANS peptide (100 μM) was also found to decrease constitutive(basal) mucus secretion by one hour incubation. Cells were treated asdescribed above except that no PMA or 8-Br-cGMP was used. In addition, anegative control peptide of the same amino acid composition as the MANSpeptide in random order (RNS; random N-terminal sequence) did not affectconstitutive mucus secretion. Results are graphed in FIG. 1B. Singleasterisk (*) indicates that the response was statistically differentthan the media control (column 1).

Example 4 UTP-Induced Mucin Secretion is Blocked by Peptide MANS

These experiments were carried out similarly to those described inExample 3. To test for stimulated secretion, the cells were exposedapically and basolaterally to uridine 5′-triphosphate (UTP) at aconcentration of 0.1 mM in media. Cells were pre-incubated for 15minutes with the MANS peptide and test cultures were then co-incubatedwith the MANS peptide and UTP for 45 minutes.

Results are shown in FIG. 2, where column 1 is the media/control; column2=0.1 mM UTP; column 3=0.1 mM UTP and 1 μM MANS peptide; column 4=0.1 mMUTP and 10 μM MANS peptide; and column 5=0.1 mM UTP and 100 μM MANSpeptide. Single asterisks (*) indicate that the measured response wasstatistically different than the media control (column 1), and doubleasterisks (**) indicate that the response was statistically differentthan that observed in stimulated cells that were not exposed to the MANSpeptide (column 2). The MANS peptide at 10 and 100 μM significantlyreduced UTP-stimulated mucus secretion.

Example 5 Effect of MA-PSD Peptide on Mucin Secretion

These experiments were carried out using normal human bronchialepithelial cells in vitro as described above. A myristoylated peptidecomposed of the 25 amino acid phosphorylation site domain of MARCKS(MA-PSD peptide; SEQ ID NO:2) was prepared. Test cells were preincubatedfor 15 minutes in apical and basolateral media containing the MA-PSDpeptide (1, 10 or 100 μM), and then co-incubated for 15 minutes with thepeptide and stimulus (100 nM PMA plus 1 μM 8-Br-cGMP). Control cellswere preincubated in media only (column 1 of FIG. 3A); or with 100 nMPMA only (column 2); or with 100 nM PMA and 1 μM 8-Br-cGMP (column 3).

Stimulation by PMA and 8-Br-cGMP caused about a 100% increase in mucussecretion over control levels. This increase was augmented in adose-dependent manner by pre- and co-incubation with the MA-PSD peptide,1 or 10 μM. Interestingly, stimulated levels of mucus secretion wereunaffected by the presence of 100 μM peptide. Results are graphed inFIG. 3A. Results using MANS peptide (1, 10 and 100 μM) are provided incolumns 7-9 for comparison. Single asterisks (*) indicate that themeasured response was statistically different than the media control(column 1), and double asterisks (**) indicate that the response wasstatistically different than that observed in stimulated cells that werenot exposed to the peptide (column 3).

The effect of MA-PSD peptide (1, 10 and 100 μM) on basal mucin secretionwas also measured. Cells as described above were incubated for one hourwith the MA-PSD peptide (no PMA or 8-Br-cGMP). Results are graphed inFIG. 3B. The single asterisk (*) indicates that the response to 100 μMof MA-PSD peptide was statistically different than the media control(column 1), whereas no statistically significant difference was seenwhen 1 or 10 μM of peptide was used (columns 3 and 4).

Example 6 Inhibition of Mucus Secretion by Antisense Oligonucleotides

Using an antisense oligonucleotide directed to the endogenous humanMARCKS gene, mucus secretion was inhibited in vitro in human airwayepithelial cells. The in vitro assay system as described in Example 1was utilized to test the effects of antisense oligonucleotides to MARCKSmRNA.

An antisense oligonucleotide was constructed by a commercial supplier(Chemicon International, Temecula, Calif.; in conjunction withBiognostik GmbH, Gottingen, Germany) based on the human MARCKS genesequence of Sakai et al. (Genomics 14:175 (1992); GenBank accessionnumber D10522, D90498). A control oligonucleotide was also constructed.

The oligonucleotides were administered to the differentiated airwayepithelial cultures by incubation in media containing theoligonucleotides (5 μM) for three days. The oligonucleotide was suppliedto the apical surface of the cells in 0.4 ml of media containinglipofectin reagent (2 μg/ml; Gibco BRL). Cells were incubated with theoligonucleotide in the presence of lipofectin for 24 hours. Following amedia change, cells were incubated with the oligonucleotide alone for anadditional 48 hours. To test the ability of the oligonucleotides toaffect stimulated mucus secretion, test cells were stimulated for 15minutes with 100 nM PMA and 1 μM 8-Br-cGMP (activators of PKC and PKG,respectively).

Results are shown in FIG. 5, where column 1=media/control; column2=cells stimulated with PMA and 8-Br-cGMP; column 3=cells exposed to 5μM control oligonucleotide and stimulated with PMA and 8-Br-cGMP; andcolumn 4=cells exposed to 5 μM antisense oligonucleotide and stimulatedwith PMA and 8-Br-cGMP. Single asterisks (*) indicate that the measuredresponse was statistically different than the media control (column 1),and double asterisks (**) indicate that the response was statisticallydifferent than that observed in stimulated cells that were not exposedto an oligonucleotide (column 2).

Mucus secreted from the airway epithelial cells after stimulation by PKCand PKG activators was assessed using an ELISA (antibody capturemethod). The control oligonucleotide (column 3) had no effect onstimulated mucus secretion. In contrast, the antisense oligonucleotide(column 4) caused a statistically significant decrease in mucussecretion compared to stimulated levels.

These results indicate that MARCKS antisense oligonucleotides inhibitstimulated mucus secretion, although a basal level of mucus secretioncan be maintained by selection of appropriate dosages. In contrast,control oligonucleotides had no effect on stimulated secretion.

Example 7 TNF-α Up-Regulates MARCKS Expression And Augments MucinHypersecretion

NHBE cells were incubated with 10 ng/ml human recombinant TNF-α ormedium alone for 4 hrs, then stimulated with PMA (100 nM)+8-Br-cGMP (1μM) for 15 min, or UTP (0.1 mM) for 2 hrs. Secreted mucin was collectedand measured by ELISA. Total RNA and protein were isolated from treatedcells. MARCKS mRNA was assessed by Northern hybridization, and proteinby Western blotting. The results are shown in FIGS. 6A, 6B and 6C.

The Northern Blot and graph shown in FIG. 6A show an increase in MARCKSmRNA in cells incubated with TNF-α (lane 2 of blot, column 2 of graph)compared to cells incubated in medium alone (lane 1 of blot, column 1 ofgraph). The Western-blot and graph shown in FIG. 6B show a three- tofour-fold increase in MARCKS protein in cells incubated with TNF-α (lane2 of blot, column 2 of graph) as compared with cells incubated withmedium only (lane 1 of blot, column 1 of graph). The graph in FIG. 6Cshows that in cells incubated with TNF-α, mucin hypersecretion wassignificantly augmented in response to subsequent stimulation byPMA+8-Br-cGMP or UTP when compared to mucin secretion of cells incubatedin medium only. Data are presented as mean±SEM (n=6 at each point).Single asterisks (*) indicate a statistically significant differencefrom control (medium-only) samples (p<0.05). Single cross marks (†)indicate a statistically significant difference from stimulus (p<0.05).

Example 8 Okadaic Acid Blocks Stimulated Mucin Hypersecretion

NHBE cells were pre-incubated with okadaic acid (500 nM) for 15 min at37° C./5% CO₂, then stimulated with PMA (100 nM)+8-Br-cGMP (1 μM) for 15min, or with UTP (0.1 mM) for 2 hours. Secreted mucin in the apicalmedium was collected and assayed by ELISA. The results are shown in FIG.7.

The graph shown in FIG. 7 shows that okadaic acid, a phosphataseinhibitor, blocks mucin hypersecretion induced by PMA+8-Br-cGMP or UTP.Column 1 shows the results of incubation with medium alone for 30 min.Column 2 shows the results of pre-incubation with medium alone for 15min, then incubation with PMA+8-Br-cGMP for an additional 15 min. Column3 shows the results of pre-incubation with okadaic acid for 15 min, thenco-incubation with PMA+8-Br-cGMP for an additional 15 min. Column 4shows the results of incubation with medium alone for 2 hrs. Column 5shows the results of pre-incubation with medium alone for 15 min, thenincubation with UTP for an additional 2 hrs. Column 6 shows the resultsof pre-incubation with okadaic acid for 15 min, then co-incubation withUTP for an additional 2 hrs. Data are presented as mean±SEM (n=6 at eachpoint). Single asterisks (*) indicate a statistically significantdifference from control (p<0.05). Single cross marks (†) indicatestatistically significant difference from stimulus (p<0.05).

Example 9 Mucin Hypersecretion Induced by UTP is Inhibited by Inhibitorsof the Mucus Secretion Signaling Pathway

NHBE cells were pre-incubated with the indicated inhibitor for 15 min,then stimulated with UTP (0.1 mM) for 2 hours. Secreted mucin in theapical medium was collected and assayed by ELISA. The results are shownin FIG. 8.

The graph of FIG. 8 shows that mucin hypersecretion induced by UTPinvolves activation of PKC and PKG. Column 1 indicates the results ofincubation with medium alone. Column 2 indicates the results ofincubation in 0.1 mM UTP. Column 3 indicates the results of incubationin 0.1 mM UTP+500 nM calphostin C (an inhibitor of PKC). Column 4indicates the results of incubation with 0.1 mM UTP+10 μMRp-8-Br-PET-cGMP (an inhibitor of PKG). Column 5 indicates the resultsof incubation with 0.1 mM UTP+50 μM LY83583 (an inhibitor of solubleguanylyl cyclase). Column 6 indicates the results of incubation with 0.1mM UTP+500 nM KT5720 (an inhibitor of PKA). Data are presented asmean±SEM (n=6 at each point). Single asterisks (*) indicate astatistically significant difference from control (p<0.05). Single crossmarks (†) indicate a statistically significant difference from UTPstimulation (p<0.05).

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A method of reducing mucus hypersecretion in a subject suffering froma medical condition in which mucus hypersecretion is a dominant clinicalfinding, the method comprising administration to the airways of saidsubject a pharmaceutical composition comprising a mucus-inhibitoryamount of a N-terminal myristoylated peptide selected from the groupconsisting of the MANS peptide and a N-terminal myristoylated peptidefragment thereof comprising at least the first 10 amino acids of theMANS peptide, wherein said MANS peptide consists of a N-terminalmyristoylated peptide of SEQ ID NO: 1, wherein said N-terminalmyristoylated peptide reduces MARCKS protein-related mucushypersecretion, and whereby mucus hypersecretion in the airways of saidsubject is reduced compared to that which would occur in the absence ofsaid peptide.
 2. The method according to claim 1, wherein saidN-terminal myristoylated peptide fragment comprises at least the first15 contiguous amino acids of the MANS peptide.
 3. The method accordingto claim 1, wherein said N-terminal myristoylated peptide fragmentcomprises at least the first 20 contiguous amino acids of the MANSpeptide.
 4. The method according to claim 1, wherein said mucus issecreted from a mucus-secreting cell that is an epithelial cellcontained within airway mucous membranes.
 5. The method according toclaim 1, wherein said condition comprises the dominant clinical findingof mucus hypersecretion in the respiratory tract.
 6. The methodaccording to claim 5, wherein said condition is selected from the groupconsisting of chronic obstructive pulmonary disease (COPD), chronicbronchitis, bronchiectasis, emphysema, cystic fibrosis, pneumonia,influenza, rhinitis, and the common cold.
 7. The method according toclaim 6, wherein said condition is selected from the group consisting ofCOPD, chronic bronchitis, and cystic fibrosis.
 8. The method accordingto claim 1, wherein said administration of the pharmaceuticalcomposition comprises administration by inhalation, topicaladministration, oral administration, rectal administration or vaginaladministration.
 9. The method according to claim 1, wherein saidadministration of the pharmaceutical composition is by inhalation. 10.The method according to claim 1, wherein said administration of thepharmaceutical composition comprises use of an aerosol comprised ofliquid particles or solid particles.
 11. The method according to claim1, wherein the pharmaceutical composition comprises the N-terminalmyristoylated peptide contained within liposomes.
 12. The methodaccording to claim 5, wherein said administration of the pharmaceuticalcomposition is to the airways of the subject.
 13. The method accordingto claim 1, wherein said subject is a mammal.
 14. The method accordingto claim 1, further comprising administration of at least one N-terminalmyristoylated peptide.
 15. The method according to claim 1, furthercomprising administration of at least one compound that inhibits orreduces the MARCKS-related secretory pathway.
 16. The method accordingto claim 1, further comprising removal of retained mucus secretions fromthe airways of said mammalian subject prior to the administration of thepharmaceutical composition.